CN111148506A

CN111148506A - Method of using Let-7b inhibitors in cosmetic and/or nutraceutical products

Info

Publication number: CN111148506A
Application number: CN201880062148.4A
Authority: CN
Inventors: V·巴尔代; C·雷梅尔米耶
Original assignee: BASF Beauty Care Solutions France SAS
Current assignee: BASF Beauty Care Solutions France SAS
Priority date: 2017-10-03
Filing date: 2018-10-02
Publication date: 2020-05-12
Also published as: FR3071742B1; WO2019069007A1; FR3071742A1

Abstract

The present invention relates to a method of using let-7b inhibitors in cosmetic and/or nutraceutical products, and in particular to the cosmetic or nutraceutical use of an aqueous extract of seabuckthorn seeds having let-7b inhibitory ability, for inhibiting let-7b expression in skin and/or mucosa to maintain or improve its biomechanical properties, in particular its firmness, elasticity and/or density.

Description

Method of using Let-7b inhibitors in cosmetic and/or nutraceutical products

The present invention relates to methods of using potential inhibitors of micrornahsa-Let-7 b-5p (hereinafter Let-7b) in the cosmetic and/or nutraceutical field.

The invention also relates to the use of novel cosmetic and/or nutraceutical ingredients and also to compositions comprising the ingredients for inhibiting the expression of Let-7b, in particular in healthy skin and/or healthy mucous membranes, and/or for maintaining and/or improving the biomechanical properties of healthy skin and/or healthy mucous membranes.

The structure and properties of the skin change under the influence of complex biological, physical and biomechanical processes, resulting in a decrease of the elasticity, firmness and density of the dermis. This results in unaesthetic sagging and drooping of body skin, especially facial skin.

From a structural point of view, the impairment of the biomechanical properties of the skin is generally caused by a change in the organization of the extracellular matrix of the dermis. These changes comprise in particular a deregulation of the synthesis of glycosaminoglycans (or GAGs), in particular of chondroitin sulphate, which is involved in the structural function and physiological regulation of the skin. These changes also include damage in the polymerization and crosslinking of collagen and elastin fibers. In this case, these fiber networks lose their biomechanical properties, and the skin loses its support function and becomes sagging.

These phenomena are caused on the one hand by intrinsic factors, in particular genetic factors, and on the other hand also by extrinsic factors, such as ultraviolet light and/or smoking and/or pollution. These latter factors can significantly lead to epigenetic modifications in which there may be regulation by micrornas.

Endogenous micrornas (or mirnas) are a class of small, non-coding RNAs, typically 20 to 24 nucleotides in length, that down-regulate gene expression at the post-transcriptional level. They link the 3' -UTR region of their target mRNA on a base-to-base basis, leading to their degradation and thus to a reduction or inhibition of translation of the corresponding protein in the cell. In the 2000 s, early studies demonstrated that these molecules act as biological regulators and are involved in many processes in living organisms. Understanding the epigenetic regulation of appearance and the discovery of miRNA-targeted mrnas have long been a significant topic, particularly in the therapeutic and cosmetic fields.

Computational algorithm-assisted sequence alignment prediction is one of the methods used for this purpose. However, experimental results show that the alignment, even if perfect, does not constitute absolute evidence of the presence of an in vivo interaction between a miRNA and its target. Indeed, since the expression of mirnas and mrnas is cell-specific, prediction by sequence alignment is hampered by the fact that: this prediction does not take into account the co-expression or absence of the two entities in the cell.

Applicants found that Let-7b epigenetically down-regulated the synthesis of five proteins in fibroblasts, fibrillarin 1(FBN1), xylosyltransferase 1(XYLT1), TGF β receptor 2(TGFBR2), integrin β 3(ITGB3), and chondroitin sulfate synthase 3(CHSY3) (see example 2).

The proteins CHSY3 and XYLT1 are responsible for the synthesis of chondroitin sulfate (such as decorin and biglycan), which is essential for the assembly of glycosaminoglycan (GAG) chains, which themselves are involved in the organization of collagen and elastic fibers within the extracellular matrix.

TGFBR2 is one of the two receptors in the TGF- β signaling pathway it plays a role in the regulation of scar formation and tissue repair, moreover, the absence of TGFBR2 expression results in a slowing of the phenomenon known as scar contracture and delays remodeling of dermal collagen within the extracellular matrix.

FNB1 is one of the major components of myofibrils, acting as weft threads within the extracellular matrix for deposition of tropoelastin during elastin synthesis.

ITGB3 forms part of an integrin complex and participates in cell-matrix interactions by binding to fibrillin 1, thereby affecting the deposition of extracellular matrix and 3D structure of dermis.

Applicants have therefore found that, in addition to down-regulating collagen synthesis in the dermis, an increase in Let-7b plays a major role in the loss of organization of the dermal extracellular matrix, and in particular in the organization of collagen fibers, elastic fibers and GAGs. The mirnas are also responsible for the reduction of firmness, elasticity and density of healthy skin and/or healthy mucous membranes.

As set forth in patent application EP 2721408, the applicant has found that molecules of the extracellular matrix (ECM) are synthesized in their native form in cells contacted with the ECM. They are secreted out of the cell from the intracellular compartment. After many maturation events, they organize and arrange themselves to form a network of ECMs. They are then in their functional form. Some cosmetic active ingredients make it possible to stimulate the synthesis of ECM collagen overall, but do not show any improvement in collagen function, i.e. neither an increase in the collagen content of the ECM nor an increase in the content of organized collagen fibres. Applicants have also found that some components improve collagen functionality, i.e. the quality of organization of collagen fibres in the ECM and/or the amount of organized collagen fibres in the ECM, but no increase in any procollagen synthesis is measured. Thus, the high synthesis of ECM molecules measured in the cell and/or extracellular matrix of cultured cells is not necessarily reflected in a greater amount of functional molecules.

The invention therefore also relates to the cosmetic and/or nutraceutical use of Let-7b inhibitors for improving the molecular functionality of the extracellular matrix. The present invention therefore relates to the use of a Let-7b inhibitor for maintaining and/or improving the organization quality of the extracellular matrix, and/or for maintaining and/or improving the amount of organized molecules of the extracellular matrix, in particular collagen fibers and/or elastic fibers, and/or for maintaining and/or increasing the synthesis of glycosaminoglycans, in particular chondroitin sulphate.

Thus, the cosmetic and/or nutraceutical use of inhibitors of Let-7b expression makes it possible to maintain and/or improve the biomechanical properties of healthy skin and/or healthy mucous membranes, preferably the firmness and/or elasticity and/or density of healthy skin and/or healthy mucous membranes, in particular of healthy dermis. r is

Such novel cosmetic targets have been identified and there is a need to identify a novel means for testing potential inhibitors.

Indeed, as described above, mirnas such as Let-7b have cell-specific expression. Thus, the applicant developed a method using potential inhibitors of Let-7b that allows for cell-specific expression of mirnas and mrnas.

By virtue of the latter, the applicant has found that aqueous extracts of seabuckthorn (Hippophae rhamnoides L.) seeds (hereinafter simply seabuckthorn) are inhibitors of Let-7b expression, and are particularly advantageous for their cosmetic and/or nutraceutical use: for maintaining and/or improving the biomechanical properties of healthy skin and/or healthy mucous membranes, in particular for maintaining and/or increasing the firmness and/or elasticity and/or density of healthy skin and/or healthy mucous membranes, in particular of healthy dermis, and/or for maintaining and/or improving the organization quality of the extracellular matrix, and/or for maintaining and/or increasing the amount of organized molecules of the extracellular matrix, in particular collagen fibers and/or elastic fibers, and/or for maintaining and/or increasing the synthesis of glycosaminoglycans, in particular chondroitin sulphate.

Hippophae rhamnoides (also called rhamnoides) is a woody plant species found in europe (especially germany) and asia (especially china) and belongs to the Elaeagnaceae (Elaeagnaceae).

The use of prunus maritima has been known in traditional Tibetan medicine for over a thousand years, and the plant is widely used in traditional Chinese medicine, especially for the treatment of cardiovascular diseases. Greeks also use the fruit of hippophae rhamnoides as a treatment for stomachache and scurvy for its analgesic properties.

Seed-derived oils are very widely used due to their high content of polyunsaturated fatty acids, in particular as anti-ageing agents in cosmetics, but also as food supplements or for healing burns in dermatology.

The production of oil from seabuckthorn seeds produces a residue consisting of the aqueous fraction of the seeds. The latter fraction is usually discarded and constitutes a significant loss of material. The applicant has found, particularly unexpectedly, that such aqueous fractions obtained from plants, which have been studied only rarely to date, have the potential to constitute many of the actives that are the subject of the present invention.

Patents FR 2840809, JP 4933768 and JP 4495925 disclose the use of a hydrophilic fraction of sea buckthorn fruits as an anti-ageing agent. However, none of these patents disclose the use of aqueous extracts of seabuckthorn seeds.

Patent application EP 2282750 describes a process for the preparation of a catalyst from supercritical CO₂Use of the residue resulting from extraction of sea buckthorn berries, further extracted by water extraction, for maintaining healthy skin, reducing skin aging, for maintaining and improving skin elasticity, or as an active ingredient for promoting skin regeneration and scar formation. However, said patent application does not disclose the cosmetic use of aqueous extracts of seabuckthorn seeds.

KR 101276141 discloses a method for extracting sea buckthorn seeds which makes it possible to remove oily compounds, comprising the use of C₁-C₄Alcohol was used as the extraction solvent, and vacuum filtration was performed using celite. The obtained extract has antioxidant and antibacterial properties, and can be used as active ingredient in composition for preventing skin aging. However, said patent does not disclose the cosmetic use of aqueous extracts of seabuckthorn seeds for inhibiting Let-7b expression to maintain and/or improve the biomechanical properties of the skin.

Thus, to the best of the applicant's knowledge, no prior art discloses the cosmetic and/or nutraceutical use of aqueous extracts of seabuckthorn seeds for maintaining and/or improving the biomechanical properties of the skin, in particular for maintaining and/or increasing the firmness and/or elasticity and/or density of the skin, in particular of the healthy dermis, and/or for maintaining and/or improving the texture quality of the extracellular matrix, and/or for maintaining and/or increasing the amount of organized molecules of the extracellular matrix, in particular collagen fibres and/or elastic fibres, and/or for maintaining and/or increasing the synthesis of glycosaminoglycans, in particular chondroitin sulphate.

In a first aspect of the invention, methods of using Let-7b inhibitors are provided.

The second aspect of the present invention also consists in providing novel cosmetic and/or nutraceutical active ingredients capable of inhibiting the expression of Let-7b, in particular in the skin.

Such ingredients have the following advantages: maintaining and/or improving the biomechanical properties of healthy skin and/or healthy mucous membranes, in particular the firmness and/or elasticity and/or density of healthy skin and/or healthy mucous membranes, in particular healthy dermis, and/or maintaining and/or improving the organization quality of the extracellular matrix, and/or maintaining and/or improving the amount of organized molecules of the extracellular matrix, in particular collagen fibers and/or elastic fibers, and/or maintaining and/or increasing the synthesis of glycosaminoglycans, in particular chondroitin sulphate.

Another aspect of the invention is also the use of the non-valuable fraction of hippophae rhamnoides, in particular in the production of cosmetic compositions.

The advantage of this novel cosmetic active ingredient is that it is topically acceptable to the skin, in particular the scalp and/or mucous membranes, it is non-toxic, readily available, and can be easily produced and packaged on an industrial scale.

A first subject of the present invention is a method for using a Let-7b inhibitor in a cosmetic and/or nutraceutical product, comprising the following steps:

a) contacting the target cell with a potential inhibitor of Let-7b,

b) the expression of Let-7b was quantified,

c) selecting the potential inhibitor of Let-7b in the event that expression of Let-7b is reduced,

d) the selected potential inhibitors of Let-7b are used as active ingredients in cosmetic and/or nutraceutical products.

For the purposes of the present invention, the term "potential inhibitor" refers to any extract of plant, animal and/or prokaryotic biological material, as well as any molecule or mixture of molecules derived from chemical synthesis and mixtures thereof. Preferably, the potential inhibitor is an extract of a plant biological material.

After the potential inhibitor contact is made, such extract and/or such synthetic molecule is selected as a "Let-7 b inhibitor" when the amount of Let-7b as measured by RT-qPCR is lower than that measured without contacting the potential inhibitor under the same or similar temperature, contact time and operating conditions. Preferably, it is at least 10%, more preferably at least 30% inhibition in the presence of a potential Let-7b inhibitor compared to the amount of Let-7b measured in the absence of a potential inhibitor according to the invention.

The term "Let-7 b" is to be understood as "has-Let-7 b-5 p". In SEQ ID NO: the sequence of this microRNA is described in 1.

Step a) corresponds to performing the contacting, i.e. adding the potential inhibitor to the target cell and/or to the medium of said target cell. Preferably, the target cell is a fibroblast, preferably a human, preferably healthy.

Advantageously, fibroblasts are those expressing Let-7 b; more preferably, these fibroblasts are derived from dermal connective tissue.

For the purposes of the present invention, the term "target cell" means all cells in which the target protein and/or its corresponding mRNA is co-expressed with Let-7 b.

For the purposes of the present invention, the expression "target protein and/or its corresponding mRNA" means all proteins whose expression is regulated by Let-7b and/or mrnas encoding these proteins preferably the protein to be regulated and/or its corresponding mRNA is fibrillin 1(FBN1) and/or xylosyltransferase 1(XYLT1) and/or TGF β receptor 2(TGFBR2) and/or integrin β 3(ITGB3) and/or chondroitin sulfate synthase 3(CHSY3), preferably xylosyltransferase 1(XYLT1) and/or integrin β 3(ITGB3) and/or chondroitin sulfate synthase 3(CHSY3), more preferably xylosyltransferase 1(XYLT1) and/or chondroitin sulfate synthase 3(CHSY 3).

Unless otherwise indicated, the term "healthy" cells, in particular "healthy" fibroblasts, means cells, in particular fibroblasts, which do not show any pathology.

The target cell is preferably a fibroblast. Advantageously, fibroblasts are those expressing Let-7 b; more preferably, these fibroblasts are derived from dermal connective tissue.

The target cells are pre-cultured according to conventional culture methods known to those skilled in the art. Preferably, in

Culture medium

Culturing the target cell.

According to an advantageous embodiment of the method of the invention, the potential inhibitor is present in a concentration range of 1X 10 by weight of the Let-7b inhibitor relative to the volume of the culture medium^-4% to 10% (w/v), preferably 0.001% to 0.1% (w/v), more preferably 0.003% to 0.02% (w/v). Advantageously, the tested concentration of potential inhibitor is between 0.003% and 0.02% (w/v) by weight of the Let-7b inhibitor relative to the total volume of the culture medium.

Preferably, the contacting is performed for a period of time in the range of 12 hours to 72 hours. Advantageously, the contacting is carried out for about 48 hours.

Advantageously, the contacting is carried out at a temperature in the range of 20 ℃ to 45 ℃, preferably at ambient temperature, preferably at about 37 ℃.

By way of example and not limitation, the cells may be cultured according to the protocol detailed in example 3.1.

Step b), called "quantification step", is a step intended to measure the expression of Let-7 b. Can be carried out using any known molecular biological measurement method. The quantification step is a direct quantification by measuring the amount of Let-7b and/or an indirect quantification by measuring the amount of proteins regulated by Let-7b and/or their corresponding mrnas.

Direct quantification may be by measuring the amount of Let-7b, in particular by measuring on an RNA chip and/or by measuring using real-time reverse transcription polymerase chain reaction (RT-qPCR or real-time RT-PCR).

Indirect quantification may also be by measuring the amount of the target protein and/or its corresponding mRNA. The amount of protein and/or its corresponding mRNA is inversely related to the expression level of Let-7 b. Quantification of the target mRNA may be performed by measurement on an RNA chip and/or by measurement using real-time reverse transcription polymerase chain reaction (RT-qPCR or real-time RT-PCR). The quantification of the target protein can be performed by Western blotting (Western blotting).

Preferably, the quantification of Let-7b expression is performed by direct quantification using RT-qPCR.

In an advantageous and non-limiting manner, step b) can be carried out according to the protocol described in example 3.2.

Step c), referred to as "selection step", comprises a comparison of the results of step b) with respect to one or more controls positive for the inhibition of Let-7b and/or with respect to one or more controls negative for the inhibition of Let-7b and/or several substances having a potential activity between them and/or with respect to the absence of any substance added to the culture medium and/or to the cells. Preferably, the comparison is made with respect to the amount measured in the absence of any substance added to the medium and/or cells.

Advantageously, in the context of the present invention, the applicant believes that the selection of potentially active molecules can be made during screening for Let-7b synthesis inhibition, which can be described as "strong". For the purposes of the present invention, the term "strongly inhibiting" means any inhibition greater than or equal to 30% of the reference activity. The reference activity is measured under the same or similar conditions of temperature, contact time and operation, without having contacted the target cell with the potentially active substance.

In a preferred embodiment of the method of use according to the invention, the Let-7b inhibitor is an aqueous extract of sea buckthorn seeds.

Step d) corresponds to the use of the selected Let-7b inhibitor as an active ingredient in a cosmetic and/or nutraceutical product.

The subject of the present invention is also a method of using a Let-7b inhibitor in a cosmetic and/or nutraceutical as described above, wherein the Let-7b inhibitor is used for maintaining and/or improving the biomechanical properties of healthy skin and/or healthy mucous membranes, in particular for maintaining and/or improving the firmness and/or elasticity and/or density of healthy skin and/or healthy mucous membranes, in particular of healthy dermis.

The subject of the present invention is also a method of using a Let-7b inhibitor in a cosmetic and/or nutraceutical as described above, wherein the Let-7b inhibitor is used to maintain and/or improve the firmness and/or density of healthy skin and/or healthy mucous membranes, in particular healthy dermis.

A subject of the present invention is also a method for using a Let-7b inhibitor in a cosmetic and/or nutraceutical product, as described above, wherein the Let-7b inhibitor is used for maintaining and/or improving the organization quality of the extracellular matrix, and/or for maintaining and/or improving the amount of organized molecules of the extracellular matrix, in particular collagen fibers and/or elastic fibers, and/or for maintaining and/or increasing the synthesis of glycosaminoglycans, in particular chondroitin sulfate.

The subject of the present invention is also the cosmetic and/or nutraceutical use of an aqueous extract of seabuckthorn seeds for inhibiting the expression of Let-7b in healthy skin and/or healthy mucous membranes and/or for maintaining and/or improving the biomechanical properties of healthy skin and/or healthy mucous membranes, in particular the firmness, elasticity and/or density of healthy skin and/or healthy mucous membranes, in particular healthy dermis.

The subject of the present invention is also a cosmetic care method comprising the application, preferably topical application, of an aqueous extract of seabuckthorn seeds and/or of a cosmetic composition comprising an aqueous extract of seabuckthorn seeds to healthy skin, in particular to healthy scalp and/or healthy mucous membranes, in order to inhibit the expression of Let-7b in healthy skin and/or healthy mucous membranes or to maintain or improve the biomechanical properties of healthy skin and/or healthy mucous membranes, in particular to maintain or improve the firmness, elasticity and/or density of healthy skin and/or healthy mucous membranes, in particular healthy dermis. The application can be carried out on all or part of a healthy body and/or the face and/or scalp, preferably on the legs, thighs, arms, abdomen, neck, armpits, whole or part of the face, preferably on the cheeks and/or angular regions of the face.

In particular, the cosmetic and/or nutraceutical use or the method is achieved to maintain or improve the organization quality of the extracellular matrix, to maintain or improve the amount of organized molecules of the extracellular matrix, in particular collagen fibers and/or elastic fibers, and/or to maintain or increase the synthesis of glycosaminoglycans, in particular chondroitin sulphate.

The extract according to the invention is a seed extract. For the purposes of the present invention, a seed corresponds to a seabuckthorn fruit (or fruit) without a flesh portion (also known as a pericarp).

The extract may be obtained by vegetable water extraction methods known in the art, for example by immersing at least a part of the seed in an amount of preferably 1 to 90% by weight (w/w), preferably 1 to 50% by weight (w/w), more preferably 5 to 20% by weight (w/w), relative to the total weight of the seed part and the solvent, in water or a solvent mixture such as water/alcohol, water/glycol or water/polyol. For example, the extracts may be obtained by extraction in a water/ethanol mixture, in particular in a ratio of 70/30 each by volume. Preferably, the extract is obtained by aqueous extraction using water as the sole solvent.

For the purposes of the present invention, the term "water extraction" means any extraction carried out with an aqueous solution comprising at least 60% by weight (w/w), advantageously at least 70% by weight (w/w), in particular at least 80% by weight (w/w), more particularly at least 90% by weight (w/w), in particular at least 95% by weight (w/w) of water, relative to the total weight of the aqueous solution. According to a preferred embodiment, the water extraction of seabuckthorn seeds is performed using water as the only solvent.

According to an advantageous embodiment, the extract may thus be obtained by aqueous extraction with seabuckthorn seeds in an amount ranging from 1% to 50% by weight (w/w), relative to the total weight of seabuckthorn seeds and solvent, preferably using water as the sole solvent.

The extract may be obtained by aqueous extraction at a temperature in the range of from 4 ℃ to 95 ℃, preferably from 20 ℃ to 95 ℃, more preferably from 50 ℃ to 95 ℃, preferably in the range of from 70 ℃ to 90 ℃. In a particular embodiment, the extraction is performed at about 80 ℃.

The extraction may be performed for a period of 1 hour to 24 hours, preferably 1 hour to 12 hours, more preferably 1 hour to 6 hours. Advantageously, the extraction is carried out for a time in the range of 2 to 3 hours.

Preferably, the extract is prepared according to the protocol described in example 1 a).

An additional step consisting in the addition of maltodextrin may be carried out. Advantageously, at least 20% by weight (w/w), preferably at least 50% by weight (w/w), more preferably at least 70% by weight (w/w) of maltodextrin is added to the extract relative to the total weight of the extract and maltodextrin. The extract may then be concentrated by evaporation of the solvent and/or dried by freeze drying and/or spray drying.

In a preferred embodiment of the invention, after the addition of maltodextrin to the solution, the extract is dried by spray drying.

In a preferred embodiment of the invention, the seabuckthorn seeds are first extracted using supercritical carbon dioxide extraction.

According to this embodiment, the aqueous extract of seabuckthorn seeds is obtained by aqueous extraction of the by-product of supercritical carbon dioxide extraction of seabuckthorn seeds.

Supercritical carbon dioxide extraction is a technique familiar to those skilled in the art which allows the separation of the oily fraction of the compound to be extracted and can therefore be used in various applications. For the purposes of the present invention, this extracted residue is called "by-product" and contains all the compounds not extracted by the technique described. Thus, it refers to the defatted fraction of seabuckthorn seeds.

For the purposes of this statement, an aqueous extract of seabuckthorn seeds contains only minor amounts of lipid compounds. Preferably, the extract comprises a concentration ranging from 1 × 10 relative to the total weight of the extract^-6% to 1% (w/w) of lipid compounds, more preferably 0.01% to 1% (w/w), and advantageously the extract contains 0.1% to 1% (w/w) of lipid compounds relative to the total weight of the extract.

This extraction technique makes it possible to obtain by-products free from residual solvents and which can be more easily separated and incorporated into cosmetic and/or nutraceutical compositions.

This technique can be carried out in the presence or absence of a co-solvent; advantageously, it is carried out in the absence of any co-solvent. It may be carried out at a pressure ranging from 50 to 80 bar and at a temperature ranging from 25 ℃ to 70 ℃; preferably, it is carried out at about 60 bar and about 30 ℃. The duration of the supercritical extraction may range from 30 to 120 minutes, preferably from 60 to 100 minutes.

The by-products are then extracted by means of water extraction as described above.

Preferably, the extract is prepared according to the protocol described in example 1 b).

Another aspect of the present invention relates to the use of a Let-7b inhibitor for maintaining or improving the biomechanical properties of healthy skin and/or healthy mucous membranes, in particular the firmness, elasticity and/or density of healthy skin and/or healthy mucous membranes, in particular healthy dermis.

Furthermore, the present invention relates to a cosmetic care method comprising the application, preferably topical application, of a Let-7b inhibitor and/or a cosmetic composition comprising a Let-7b inhibitor to healthy skin, in particular to healthy scalp and/or healthy mucous membranes, in order to maintain or improve the biomechanical properties of the healthy skin and/or healthy mucous membranes, in particular the firmness, elasticity and/or density of the healthy skin and/or healthy mucous membranes, in particular of the healthy dermis. The application can be carried out on all or part of a healthy body and/or the face and/or scalp, preferably on the legs, thighs, arms, abdomen, neck, armpits, whole or part of the face, preferably on the cheeks and/or angular regions of the face.

Preferably, the methods and uses are effected to maintain or improve the organization quality of the extracellular matrix, to maintain or increase the amount of organized molecules of the extracellular matrix, in particular collagen fibers and/or elastic fibers, and/or to maintain or increase the synthesis of glycosaminoglycans, in particular chondroitin sulfate.

In a particular embodiment, the Let-7b inhibitor is selected by a method according to the invention, as described above. Many Let-7b inhibitors are available to those skilled in the art. For example, oligonucleotides complementary to the Let-7b sequence, such as the sequence SEQ ID NO: 2.

The present invention relates to cosmetic compositions comprising a Let-7b inhibitor and at least one cosmetically acceptable excipient.

In one embodiment of the present invention, a cosmetic treatment method comprises:

a) identifying healthy skin areas where it is desired to inhibit the expression of Let-7b in healthy skin and/or healthy mucous membranes, or to maintain or improve the biomechanical properties of healthy skin and/or healthy mucous membranes, in particular the firmness, elasticity and/or density of healthy skin and/or healthy mucous membranes, especially healthy dermis; and

b) applying topically to the area of skin a seabuckthorn extract according to the invention, preferably a cosmetic composition comprising a seabuckthorn extract according to the invention.

In an alternative embodiment of the present invention, a cosmetic treatment method comprises:

a) identifying healthy skin areas showing a non-pathological loss of firmness, elasticity and/or density of healthy skin and/or healthy mucous membranes, in particular healthy dermis; and

For the purposes of the present invention, the expression "cosmetic use and/or cosmetic composition" means a use and/or composition that is non-pharmaceutical, i.e. does not require therapeutic treatment, i.e. is intended for use on healthy skin, in particular on any area of healthy scalp and/or healthy mucous membrane.

For the purposes of the present invention, the expression "nutraceutical use and/or nutraceutical composition" means a use and/or composition which is not administered orally, i.e. does not require therapeutic treatment.

The term "healthy skin" and/or "healthy mucous membranes" and/or "healthy scalp" means the area of skin and/or mucous membranes and/or scalp to which the extract according to the invention is applied and which is called "non-pathological" by the dermatologist, i.e. the area of skin and/or mucous membranes and/or scalp which does not present any infections, scars, diseases, wounds or lesions and/or other types of skin diseases.

The term "mucosa" means the ocular, vaginal, urogenital, anal, nasal and/or buccal mucosa, the labial and/or gingival mucosa, preferably the ocular and/or buccal mucosa.

For the purposes of the present invention, the expression "inhibiting the expression of Let-7 b" means reducing the amount of Let-7b, in particular in healthy skin and/or healthy mucous membranes, preferably in healthy dermis. Preferably, it is at least 10%, more preferably at least 30% inhibition in the presence of an aqueous extract of seabuckthorn seeds relative to the amount of Let-7b measured in the absence of the extract according to the invention.

A particular embodiment of the present invention relates to the use of an aqueous extract of hippophae rhamnoides seed according to the invention for inhibiting the expression of Let-7b in healthy dermis and/or healthy basal layer, in particular in healthy dermoepidermal junction, preferably in healthy dermis.

A preferred embodiment of the present invention relates to the use of an aqueous extract of hippophae rhamnoides seed according to the invention for inhibiting the expression of Let-7b in healthy fibroblasts.

The amount of Let-7b can be measured using any known molecular biological quantification method. It may be a direct quantification of the amount of Let-7b miRNA, in particular by measurement on an RNA chip and/or by measurement of real-time reverse transcription polymerase chain reaction (RT-qPCR or real-time RT-PCR). Preferably, the amount of Let-7b is measured by RT-qPCR according to the usual methods known to the person skilled in the art. More preferably, the measurements are performed according to example 3.2.

For the purposes of the present invention, the expression "biomechanical properties of healthy skin and/or healthy mucous membranes" means the firmness and/or elasticity and/or density and/or resistance to compression and/or resistance to stretching and/or flexibility and/or extensibility and/or ability to withstand deformation of healthy skin and/or healthy mucous membranes, in particular healthy dermis. Preferably, it relates to the firmness and/or elasticity and/or density of healthy skin and/or healthy mucous membranes, in particular healthy dermis.

The biomechanical properties of healthy skin and/or healthy mucous membranes can be studied by measurement techniques known to the person skilled in the art, in particular by traction, torsion, aspiration, indentation, levatometry, elastometry (ballistometry), by high resolution ultrasound imaging or elastography, preferably by indentation or by high resolution ultrasound imaging. Advantageously, the biomechanical properties of healthy skin and/or healthy mucous membranes were studied according to the protocol described in example 5.

For the purposes of the present invention, the expression "maintaining the biomechanical properties of healthy skin and/or healthy mucous membranes" means preventing a reduction in the biomechanical properties of healthy skin and/or healthy mucous membranes, in particular the firmness and/or elasticity and/or density of healthy skin and/or healthy mucous membranes, in particular of healthy dermis, compared to the biomechanical properties of healthy skin and/or healthy mucous membranes measured in the absence of the extract according to the present invention.

For the purposes of the present invention, the expression "improving the biomechanical properties of healthy skin and/or healthy mucosa" means that the biomechanical properties of healthy skin and/or healthy mucosa, in particular the firmness and/or elasticity and/or density of healthy skin and/or healthy mucosa, in particular of healthy dermis, are increased, preferably by at least 10%, in the presence of a Let-7b inhibitor or an aqueous extract of hippophae rhamnoides seed, compared to the biomechanical properties of healthy skin and/or healthy mucosa measured in the absence of a Let-7b inhibitor or extract according to the present invention.

According to one particular embodiment, the subject of the present invention is the cosmetic and/or nutraceutical use of the Let-7b inhibitor or the aqueous extract of seabuckthorn seeds according to the invention for maintaining and/or increasing the firmness and/or density of healthy skin and/or healthy mucous membranes, in particular healthy dermis.

In particular, the maintenance and/or improvement of the biomechanical properties of healthy skin makes it possible to limit and/or reduce the sagging of healthy skin of the face, in particular of the cheeks and/or of the angular regions of the face (commonly known as the facial curve or the facial V-zone).

Another subject of the present invention relates to the cosmetic and/or nutraceutical use of the Let-7b inhibitor or the aqueous extract of sea buckthorn seeds according to the invention for maintaining and/or improving the texture quality of the extracellular matrix, in particular of collagen fibres and/or elastic fibres.

For the purposes of the present invention, the expression "organization quality of the extracellular matrix" means the level of functional organization of the molecules constituting the extracellular matrix. As set forth in the introduction of the present patent application, the proteins FBN1 and/or XYLT1 and/or TGFBR2 and/or ITGB3 and/or CHSY3 are involved in the deposition and/or organization of the extracellular matrix and are down-regulated by Let-7 b. The inhibition of Let-7b leads to a reduced inhibition of these proteins and makes it possible to maintain and/or improve the quality of organization of the extracellular matrix.

For the purposes of the present invention, the expression "maintaining the quality of organization of the extracellular matrix" means preventing a decrease in the organization of the extracellular matrix with respect to that observed visually in the absence of the Let-7b inhibitor or extract according to the invention, in particular by limiting the down-regulation of the proteins FBN1 and/or XYLT1 and/or TGFBR2 and/or ITGB3 and/or CHSY 3.

For the purposes of the present invention, the expression "improving the quality of organization of the extracellular matrix" means improving the organization of the extracellular matrix with respect to the organization of the extracellular matrix visualized in the absence of the Let-7b inhibitor or extract according to the invention, in particular by limiting the down-regulation of the proteins FBN1 and/or XYLT1 and/or TGFBR2 and/or ITGB3 and/or CHSY 3.

The visualization of the functional organization of the extracellular matrix can be carried out by techniques of chemical techniques of immunological organization or according to the methods described in the applicant's patent EP 2721408.

Another subject of the present invention relates to the cosmetic and/or nutraceutical use of the Let-7b inhibitor or the aqueous extract of seabuckthorn seeds according to the invention for maintaining and/or increasing the amount of organized molecules of the extracellular matrix, in particular collagen fibres and/or elastic fibres.

For the purposes of the present invention, the expression "amount of organized molecules of the extracellular matrix" means the number of molecules of the extracellular matrix that are arranged and functional within the extracellular matrix.

For the purposes of the present invention, the expression "maintaining the amount of organized molecules of the extracellular matrix" means preventing a decrease in the amount of organized molecules of the extracellular matrix relative to the amount of organized molecules of the extracellular matrix measured in the absence of the Let-7b inhibitor or extract according to the invention.

For the purposes of the present invention, the expression "increasing the amount of organized molecules of the extracellular matrix" means that the amount of organized molecules of the extracellular matrix is increased relative to the amount of organized molecules of the extracellular matrix measured in the absence of the Let-7b inhibitor or extract according to the invention.

The amount of organized molecules of extracellular matrix, in particular collagen fibres and/or elastic fibres, can be measured according to the method described in the applicant's patent EP 2721408.

Another subject of the present invention relates to the cosmetic and/or nutraceutical use of the Let-7b inhibitors or aqueous extracts of sea buckthorn seeds according to the invention for maintaining and/or increasing the synthesis of glycosaminoglycans, in particular chondroitin sulphate.

For the purposes of the present invention, the expression "maintaining the synthesis of glycosaminoglycans, in particular chondroitin sulfate" means preventing a reduction in the level of synthesis of glycosaminoglycans, in particular chondroitin sulfate, relative to the level of synthesis of glycosaminoglycans, in particular chondroitin sulfate, detected in the absence of the Let-7b inhibitor or extract according to the invention.

For the purposes of the present invention, the expression "increasing the synthesis of glycosaminoglycans, in particular chondroitin sulfate", means that the level of synthesis of glycosaminoglycans, in particular chondroitin sulfate, in the presence of a Let-7b inhibitor or a seabuckthorn seed extract is increased, preferably by at least 10%, preferably by at least 50%, more preferably by at least 70%, relative to the level of synthesis of glycosaminoglycans, in particular chondroitin sulfate, detected in the absence of a Let-7b inhibitor or extract according to the present invention.

According to a preferred embodiment of the invention, the aqueous extract of seabuckthorn seeds is a topically acceptable cosmetic extract.

According to another preferred embodiment of the invention, the Let-7b inhibitor is topically acceptable.

For the purposes of the present invention, the term "topically acceptable" means suitable for topical application, non-toxic, non-irritating to healthy skin, especially healthy scalp and/or healthy mucous membranes, not causing any allergic reactions, and not being chemically unstable.

The Let-7b inhibitor or extract according to the invention is applied to healthy skin, which may be the body and/or the face and/or the scalp, preferably the legs, thighs, arms, abdomen, neck print, neck, axilla, more preferably the whole or part of the face, preferably the whole or part of the healthy skin and/or healthy mucous membranes of the cheeks and/or angular regions of the face (commonly referred to as the facial curve or facial V-zone).

The Let-7b inhibitor or aqueous extract of sea buckthorn seeds can be used alone as a cosmetic and/or nutraceutical active ingredient or can be included in a cosmetic and/or nutraceutical composition.

When used alone as active ingredient, the extract according to the invention is preferably in powder form and dissolved in a solvent, in particular a polar solvent such as water, an alcohol, a polyol, a glycol or mixtures thereof, in the presence or absence of glycerol.

In a particular embodiment of the invention, the extract according to the invention is mixed with maltodextrin. In this case, maltodextrin makes it possible to obtain cosmetic and/or nutraceutical ingredients that are not hygroscopic.

Advantageously, at least 20% by weight (w/w), preferably at least 50% by weight (w/w), more preferably at least 70% by weight (w/w) of maltodextrin is added to the extract relative to the total weight of the extract and maltodextrin. The extract may then be concentrated by evaporation of the solvent or dried, for example by freeze drying or spray drying. Preferably, the extract is dried by spray drying.

In this case advantageously the cosmetic ingredients are as described in example 6.

The subject of the present invention is also the cosmetic use of the extract according to the invention, for topical application to healthy skin, in particular to the scalp and/or mucous membranes, in a cosmetic composition comprising at least one cosmetically acceptable excipient.

The subject of the present invention is also the use of an aqueous extract of seabuckthorn seeds in a cosmetic and/or nutraceutical composition comprising at least one cosmetically acceptable excipient and in which the extract is present in a concentration range of 1 x 10 by weight of the extract according to the invention relative to the total volume of the composition^-4% to 10% (w/v), preferably 1X 10^-4% to 5% (w/v), more advantageously 1X 10^-3% to 0.5% (w/v).

In a particular embodiment of the invention, the cosmetic composition contains 0.2% by weight (w/v) of an aqueous extract of seabuckthorn seeds relative to the total volume of the composition.

Preferably, the extract is used in a composition as described in example 7.

In one embodiment of the invention, the extract is used in a cosmetic and/or nutraceutical composition for inhibiting the expression of Let-7b and/or for maintaining and/or improving the biomechanical properties of healthy skin and/or healthy mucous membranes, in particular for maintaining and/or improving the firmness and/or elasticity and/or density of healthy skin and/or healthy mucous membranes, in particular healthy dermis, and/or for maintaining or improving the organization quality of extracellular matrix, for maintaining or increasing the amount of organized molecules of extracellular matrix, in particular collagen fibers and/or elastic fibers, and/or for maintaining or increasing the synthesis of glycosaminoglycans, in particular chondroitin sulphate.

Cosmetic compositions according to the present invention may also comprise a cosmetically acceptable vehicle. The term "cosmetically acceptable vehicle" means a vehicle suitable for use in contact with human skin cells, particularly epidermal cells, without undue toxicity, irritation, or allergic response and the like, and is commensurate with a reasonable benefit/risk ratio. The vehicle may be, for example, maltodextrin.

The composition according to the invention can be administered topically and/or orally. Advantageously, topical application is carried out.

The cosmetic composition may also contain one or more cosmetically acceptable excipients selected from surfactants and/or emulsifiers, preservatives, buffers, chelating agents, denaturants, opacifiers, pH adjusters, reducing agents, stabilizers, thickeners, gelling agents, film forming polymers, fillers, matting agents, shine agents, pigments, colorants, fragrances and mixtures thereof. PCPC (personal care products committee) describes various cosmetic excipients suitable for use in the present invention.

Advantageously, the one or more excipients are selected from: polyglycerols, esters, cellulose polymers and derivatives, lanolin derivatives, phospholipids, lactoferrin, lactoperoxidase, sucrose-based stabilizers, vitamin E and its derivatives, xanthan gum, natural and synthetic waxes, vegetable oils, triglycerides, unsaponifiables, phytosterols, silicones, protein hydrolysates, betaines, amino oxides, plant extracts, sucrose esters, titanium dioxide, glycine and p-hydroxybenzoate, more preferably: steareth-2, steareth-21, ethylene glycol-15 stearyl ether, cetostearyl alcohol, phenoxyethanol, methyl paraben, ethyl paraben, propyl paraben, butyl paraben, butylene glycol, capryl glycol, natural tocopherol, glycerin, sodium dihydroxycetyl phosphate, isopropyl hydroxyketone ether, ethylene glycol stearate, triisononyl amine, octyl cocoate, polyacrylamide, isoparaffin, laureth-7, carbomer, propylene glycol, hexylene glycol, glycerin, bisabolol, dimethicone, sodium hydroxide, PEG 30-dipolyhydroxystearate, caprylic/capric triglyceride, octyl cetostearyl alcohol, dibutyl adipate, grape seed oil, jojoba oil, magnesium sulfate, EDTA, cyclomethicone, xanthan gum, citric acid, sodium lauryl sulfate, Mineral waxes and oils, isostearyl isostearate, propylene glycol dinonate, propylene glycol isostearate, PEG 8, beeswax, glyceryl esters from hydrogenated palm kernel oil, lanolin oil, sesame oil, cetyl lactate, lanolin alcohol, castor oil, titanium dioxide, lactose, sucrose, low density polyethylene, isotonic saline solution, and mixtures thereof.

The cosmetic composition according to the invention may be selected from: an aqueous solution, cream or hydrogel (especially shower gel), milk, emulsion, microemulsion or nanoemulsion (especially oil-in-water or water-in-oil based or multi-based or silicone based), mask, essence, lotion, liquid soap, ointment, mousse, patch, preferably cream, milk, emulsion, mask or essence.

The compositions used according to the invention may also contain cosmetic active ingredients, such as anti-ageing actives, which produce a complementary or synergistic effect. Among these agents, mention may be made of agents which stimulate the synthesis of dermal macromolecules or prevent their degradation, agents which stimulate the proliferation of keratinocytes, sedatives, moisturizers or agents which act on the regulation of pore size and/or open pores.

Among the anti-ageing agents, mention may be made of:

active agents for stimulating fibronectin synthesis, in particular corn extracts (such extracts are in particular sold under the name Deliner by BASF Beauty Care Solutions France (BASF Beauty Care Solutions France)^TMSold) and under the trade name of Sedama, Inc. (Sedama)

Palmitoyl pentapeptide sold;

active agents for stimulating collagen fibril formation, such as the one sold under the name Dermagenist by the Applicant^TMMarketed marjoram (Origanum majorana) extract;

active agents for stimulating the expression of leucosin and dystrophin glycans (dystoglycan) in the extracellular matrix and/or epithelial basement membrane, such as for example Perlaura, tradename, by basf beauty care solution, france^TMPolygonum bistorta (Polygonum bistorta) extract sold;

active agents for protecting the extracellular matrix fibroblast growth factor (FGF2) from degradation and/or denaturation, in particular as described in the patent application filed under number FR 0654316 under the name of the basf beauty care solution french company and sold under the trade name of the lineactor by the basf beauty care solution french company^TMSaleExtract of abelmoschus esculentus (Hibiscus abelmoscus); and/or agents for stimulating fibroblast growth, e.g. the so-called phytokinine^TMAnd marketed by basf beauty care solution french company, also in patent application EP 1119344B 1 (french company laboratories), a fermented soy extract containing peptides is described; and preferably a combination of these two extracts;

active agents for stimulating laminin synthesis, in particular malt extract modified by biotechnology, such extract being in particular under the trade name basiline by the french company of the basf beauty care solution^TMSelling;

an agent for stimulating the expression and/or activity of hyaluronan synthase 2(HAS2), such as a plant extract as described in patent application FR 2893252 a1, and in particular under the name Hyalufix by the french company of the basf beauty care solution^TMWater extracts of galangal (Alpinia galanga)) sold;

active agents for stimulating the synthesis of lysyl oxidase-like protein (LOXL), such as the extract of Aildehydes cordifolia (Geophilalacordifolia) and those described in patent application FR 2855968, in particular under the trade name Lys' last by the French company, the Pasteur beauty Care solution^TMA sold dill extract;

active agents for stimulating intracellular ATP synthesis, in particular under the trade name Seanergilium by the french company of the basf beauty care solution^TMLaminaria digitata (Laminaria digitata) algae extract for sale;

-an active agent for stimulating glycosaminoglycan synthesis, such as milk fermentation products;

-an active agent for stimulating collagen, such as retinol and/or vitamin C;

active agents such as retinoids and derivatives, oligopeptides and lipopeptides, lipoaminoacids, available under the trade name argenyl from basfbauty Care Solutions France (basfbauty Care Solutions France SAS) for inhibiting metalloproteinases (MMPs), such as more specifically MMP1, MMP2, MMP3 and MMP9^TMCommercial AcaciaExtract of leaves of trees (Arganiaspinoso); lycopene; isoflavones, quercetin, kaempferol, apigenin;

active agents for increasing LOX expression, increasing epidermal structure, such as LOX-AGE, available under the trade name of basf beauty care solution, france^TMChicory (Cichorium intybus) extract sold;

-an agent for increasing the deglycation of collagen and/or increasing the expression of type I collagen, such as the CollRepiair brand by the French company of the beauty Care solution of Pasteur^TMA combination of sold Salvia miltiorrhiza (Salvia militirhiza) leaf extract and niacin;

active agents for stimulating the synthesis of basement membrane polysaccharides and collagen, such as those sold under the name Dermican by the French company of the Beffy beauty Care solution^TMThe synthetic acetyl tetrapeptide Gln Asp Val His His sold and described in patent application WO 2005/120554 a 1;

active agents for protecting and stimulating elastin and collagen, such as under the trade name elescan by the french company of the basf beauty care solution^TMCommercially available leaf extract of Murraya paniculata (Manilkara multinervis) under the trade name Eperuline by the French company, Perffir beauty Care solution^TMA root extract of falcate wood (Eperufalcata) is sold.

As sedatives which can be preferably used in the compositions according to the invention, mention may be made of pentacyclic triterpenes, ursolic acid and its salts, oleanolic acid and its salts, betulinic acid and its salts, salicylates (and in particular zinc salicylate), bisabolol, allantoin, omega-3 unsaturated oils, cortisone, hydrocortisone, indomethacin and betamethasone, anti-inflammatory active agents, and in particular those described in patent application FR 2847267, in particular under the trade name SAS by the France beauty Care solution France, Basff

Root extract of Pueraria lobata (Pueraria lobata) and Theobroma cacao (Theobroma cacao) are sold.

In influencing the regulation of pore size and/or hairAmong the active agents with open pores, mention will be made, for example, of those sold under the name LOX-AGE by the French company of the cosmetic care solution of Pasteur^TMAmong the chicory extracts sold, and/or among the agents that influence sebum production, mention will be made, for example, of those sold under the name Mat-XS^TMSynthetic sarcosine sold by Clinical and/or under the name of Mat-XS under the name of French company, a cosmetology care solution, as described in patent application WO2010/063674^TMOrthosiphon stamineus (Orthospon stamineus) extract sold by Bright.

As moisturizer that can be preferably used in the composition according to the invention, mention may be made of a combination of pullulan, sodium hyaluronate and sodium alginate, as sold under the trade name Patch2O by French corporation of the cosmetic Care solution of Pasteur^TMThe product for sale.

Other objects, features and advantages of the present invention will become apparent to those skilled in the art upon a reading of the explanatory description, which refers to an embodiment given by way of illustration only, and should not limit the scope of the invention in any way.

The examples form an integral part of the invention and any feature which appears to be novel in any way relative to any prior art in view of its function and general nature as an integral part of the specification, including the examples. Thus, each embodiment has a general scope.

Also, in the examples, all percentages are by weight unless otherwise indicated, temperatures are in degrees celsius unless otherwise indicated, and pressures are atmospheric unless otherwise indicated.

Example 1: preparation of aqueous extract of sea buckthorn seeds according to the present invention

Example 1a) Water extraction of seabuckthorn seeds according to the invention

Seabuckthorn seeds (origin: germany) at 20% by weight relative to the total weight of seeds and water were ground with an electric grinder, then homogenized with a homogenizer for a few seconds, and then extracted with water as the only solvent at room temperature for a period of 2 hours. The extract was then centrifuged and the precipitate discarded. The obtained extract is then filtered through a 0.1-0.3 micron filter, followed by freeze-drying.

Example 1b) supercritical extraction of seabuckthorn seeds according to the invention

10% by weight of sea-buckthorn seeds (origin: Germany) were ground with an electric grinder and then extracted by supercritical carbon dioxide extraction at 60 bar, 30 ℃ for 80 minutes.

Placing the byproduct of supercritical carbon dioxide extraction of fructus Hippophae in osmotic water, stirring and maintaining at 80 + -2 deg.C for 1 hr, and adjusting pH to 5.4 + -0.2. The mixture was then cooled to 23 ℃. + -. 3 ℃. The extract was then coarse screened, then centrifuged, and the supernatant discarded. The extract is finally filtered to 0.1-0.3 micron. Maltodextrin is added in an amount ranging from 60% to 80% (w/w) relative to the total weight of the mixture. The extract obtained is then filtered again and subsequently atomized at a temperature of 180 ℃.

Example 2: determination of Let-7b target protein

The concentration changes of the five identified proteins potentially regulated by Let-7b were measured by western blotting with the aid of function gain/loss experiments.

Total protein was extracted for 72 and 96 hours after transfection of synthetic complementary RNA of Let-7b (reference numbers 4100945-.

In acrylamide gels (NuPAGE Novex 4-12% BBos-Tris)

Invitrogen corporation

) 10. mu.g of protein was separated and then transferred to a nitrocellulose membrane (general electric)

) The above. Use of Primary antibody (from St. Cruis Biotech Ltd.)

From Saimer Feishel scientific Co., Ltd, and the anti-XYLT 1, anti-CHSY 3 and anti-TGFBR 2

anti-FBN 1 and a peptide derived from

anti-ITGB 3) was used to specifically detect proteins. anti-HRP-b-actin antibody (Sigma) was used

) Controls were performed. By means of data processing software (

Gel analyzer algorithm) measures the signal intensity obtained for each strip.

Table 1: analysis of results of Western blots with and without transfection with Let-7b inhibitor

Table 2: results of Western blot analysis with or without transfection of RNA mimicking Let-7b activity.

Conclusion

Let-7b mirnas have a negative impact on the synthesis of FBN1, XYLT1, TGFBR2, ITGB3 and CHSY3, especially XYLT1 and CHSY 3.

Example 3: quantification of Let-7b in the Presence or absence of a potentially active substance

3.1 cultivation

Fibroblasts were isolated from healthy dermis by trypsinization (abdomen, women between 53 and 68 years of age) and cultured in a medium containing 10% (v/v) fetal bovine serum and antibiotics

Culture medium

Culturing in a medium and then culturing in 5% (v/v) CO₂Is incubated at 37 ℃ for 24 hours. The cells are then placed in an environment with or without the presence of potentially active substances, in the absence of any foetal calf serum in 5% CO₂Has two different concentrations of 0.003% (w/v) and 0.02% (w/v) by weight of the extract according to the invention relative to the total volume of the culture medium, for 48 hours.

3.2 quantification

Using nucleic acid isolation kit (NucleoSpin)

-Macherey-

Total RNA was extracted from various cultures (cultures were performed with or without the presence of active substances as presented in example 1b, as presented in example 3.1), with a physical separation between long and short RNAs. Then, the measurement of optical densities at 260nm and 280nm allowed the quality and quantity of RNA to be verified.

The reverse transcription kit (MiScript II RT kit) was then used

Kaijie

) The reverse transcription step was performed with 1. mu.g of short RNA from various assay conditions, and the final volume was 20. mu.l. The reaction mixture was incubated at 37 ℃ for 1 hour, then reverse transcriptase was inactivated by raising the temperature to 95 ℃ and holding for 5 minutes, and the reaction mixture was stored at-20 ℃ until assayed by quantitative PCR analysis.

The quantitative PCR step was performed using three types of primers (as presented in the table below). The first primer allows the amplification of Let-7b miRNA and the other two primers are used to amplify small nuclear RNA in order to construct a calibration curve by the delta-delta-Ct method. The step is in a thermal cycler

In (1) mu.l of a 1/250 diluted reverse transcription reaction mixture using specific oligonucleotides and a quantitative PCR kit. Each assay was performed in duplicate. After each amplification cycle, the fluorescence of SYBR Green contained in the kit was measured. Fusion curves were generated after each qPCR.

hsa-let-7b-5p forward primer TGAGGTAGTAGGTTGTGTGGTT SEQ ID NO: 3

snoRNA U6 forward primer ATGTTATGATGATGGGCGAAATGT SEQ ID NO: 4

snRNA U1A forward primer TTACCTGGCAGGGGAGATAC SEQ ID NO: 5

Poly T reverse primer TTTTTTTTTTTTTTT SEQ ID NO: 6

Table 3: reduction of the concentration of Let-7b in human fibroblasts in the presence of an extract according to the invention

Conclusion

The extract according to the invention resulted in an 18% and 39% reduction in expression of Let-7b relative to untreated controls for respective concentrations of 0.003% (w/v) and 0.02% (w/v) by weight of the extract according to the invention relative to the total volume of the composition in the culture medium.

Example 4: dermal biomassIn vitro assessment of improvement of chemical Properties

The density and firmness of the dermis were measured by means of low pressure indentation and relaxation tests on the dermis replacement.

The dermal substitute used corresponds to the applicant' s

Provided is a technique. To produce a dermal substitute, 500000 cells/cm were added²Human primary fibroblasts (from the dermis of the breast from an 18 year old female donor) seeded in DMEM/HAM F12 supplemented with 20% fetal bovine serum

Collagen sponge (from the applicant)

) The above. The medium was replaced with 50. mu.g/ml ascorbic acid daily. From the third day of culture, the dermis was treated or not with 0.02% (w/v) of the aqueous extract of the in vitro seed according to example 1b), by weight of the extract according to the invention with respect to the total volume of the composition. The culture of treated and untreated dermal substitutes was stopped after 28 days.

The dermal substitute thus obtained was then subjected to low pressure indentation and relaxation tests. The test was repeated 3 times for each sample until a constant penetration depth (100 μm) was reached and a motion was applied at a constant indentation speed (δ 1/450 μm/s for 120 seconds). Data was collected at a sampling rate of 1 kHz.

The indenter used was a spherical PTFE indenter with a radius of curvature R1/4 of 1.6 mm. Tests were conducted in air-conditioned rooms, temperature controlled at 22 ℃ to 24 ℃ and relative humidity of about 30% to 40%.

Conclusion

After the treatment, the applicant observed that the extract according to the invention resulted in a 14% increase in young's modulus (p ═ 0.06) relative to the untreated control, which demonstrates that the extract caused an increase in the biomechanical properties of the dermis.

Example 5: in vivo research on human volunteersIs especially suitable for the treatment of diabetes

A study on human volunteers was carried out in order to test the efficacy of the formulation containing 0.2% (w/v) of the extract described in example 1b with respect to the main signs of loss of the biomechanical properties of the dermis, in particular with respect to skin laxity.

1.Study protocol

27 white women, between 55 and 70 years of age, who exhibited a loss of skin firmness and elasticity, received a double-blind randomized trial comprising the formulation described in the examples on one half of the face and placebo on the other half of the face, twice daily for 56 days. Under dermatological control, results were measured at the start of treatment (D0), after 28 days (D28) and after 56 days (D56).

2.Study of changes in dermal density and elasticity

The change in dermal density and elasticity was measured using a high resolution ultrasound scanner (Dub SkinScanner-TPM, 22MHz) measuring a hyperechoic density factor, which corresponds to the percentage of pixels measured to have high intensity (between 200 and 255).

Conclusion

The applicant observed that the extract according to the invention resulted in a significant increase of the hyperechoic density factor by about 26% after 56 days of administration with respect to placebo.

3.Study of changes in skin firmness

Using means allowing measurement of skin firmness

Device

Changes in skin firmness were measured at the lower corners of the face. An increase in this factor corresponds to an increase in skin firmness.

Conclusion

The applicant observed that the extract according to the invention caused a significant increase in the firmness of the cheek skin by about 18.5% after 56 days of administration with respect to placebo, which means that the composition comprising the extract according to the invention increased the density and elasticity of the dermis.

Example 6: cosmetic composition containing aqueous extract of sea buckthorn seed

Aqueous extract of sea buckthorn seeds obtained according to example 1b)	30% by weight
		Maltodextrin	70 percent by weight

Example 7: cosmetic composition comprising aqueous extract of sea buckthorn seed

The following phases A, B, C and D were mixed together using methods known to those skilled in the art to prepare compositions according to the present invention. The ratios are expressed as percentages.

Phase A

Caprylic propyl heptyl ester 4.00

Carbonic acid dioctyl ester 4.00

Polyacrylamide sodium 0.70

Phase B

Phase C

Sclerotium gum 2.00

Phase D

Cosmetic ingredient according to example 6 0.20

And 2.00 percent of water.

Claims

1. Use of an aqueous extract of seabuckthorn seeds for cosmetic and/or nutraceutical use for inhibiting the expression of Let-7b in healthy skin and/or healthy mucous membranes or for maintaining or improving the biomechanical properties of healthy skin and/or healthy mucous membranes, in particular the firmness, elasticity and/or density of healthy skin and/or healthy mucous membranes, especially healthy dermis.

2. Use according to claim 1, for inhibiting the expression of Let-7b in healthy dermis and/or healthy basal layer, in particular in healthy dermoepidermal junction, preferably in healthy dermis.

3. Use according to any one of claims 1 and 2, for inhibiting the expression of Let-7b in healthy fibroblasts.

4. Use according to any one of claims 1 to 3, for maintaining or improving the organization quality of the extracellular matrix, for maintaining or increasing the amount of organized molecules of the extracellular matrix, in particular collagen fibres and/or elastic fibres, and/or for maintaining or increasing the synthesis of glycosaminoglycans, in particular chondroitin sulphate.

5. The use as claimed in any one of claims 1 to 4, wherein the aqueous extract of seabuckthorn seeds is obtained by aqueous extraction of a by-product of supercritical carbon dioxide extraction of seabuckthorn seeds.

6. The use as claimed in any one of claims 1 to 5, wherein the water extraction of the seabuckthorn seeds is carried out with a solvent comprising at least 60% by weight, advantageously at least 70% by weight, in particular at least 80% by weight, more particularly at least 90% by weight and in particular at least 95% by weight of water relative to the total weight of aqueous solution.

7. The use as claimed in any one of claims 1 to 6, wherein the water extraction of the seabuckthorn seeds is carried out using water as the only solvent.

8. Use of an extract according to any one of claims 1 to 7, wherein the Hippophae rhamnoides extract is present in a cosmetic and/or nutraceutical composition comprising at least one cosmetically acceptable excipient, and wherein the extract is present in a concentration range of 1 x 10 by weight of the extract according to the invention relative to the total volume of the composition^-4% to 10% (w/v), preferably 1X 10^-4% to 5% (w/v), more advantageously 1X 10, by weight of the extract according to the invention relative to the total volume of the composition^-3% to 0.5% (w/v).

9. Cosmetic treatment process comprising the application, preferably topical application, of an aqueous extract of seabuckthorn seeds as claimed in any one of claims 1 to 8 and/or of a cosmetic composition comprising the same, to healthy skin, in particular to healthy scalp and/or healthy mucous membranes, to inhibit the expression of Let-7b in healthy skin and/or healthy mucous membranes, or maintaining or improving the biomechanical properties of healthy skin and/or healthy mucous membranes, in particular maintaining or improving the firmness, elasticity and/or density of healthy skin and/or healthy mucous membranes, in particular healthy dermis, preferably maintaining or improving the texture quality of the extracellular matrix, maintaining or improving the texture of collagen fibers and/or elastic fibers, and/or increasing the synthesis of glycosaminoglycans, in particular chondroitin sulfate.

10. Cosmetic care method according to claim 9, wherein the application is carried out on all or part of the healthy body and/or face and/or scalp, preferably on the legs, thighs, arms, abdomen, neck, armpits, whole or part of the face, preferably on the cheeks and/or angular regions of the face.

11. A method of using a Let-7b inhibitor in a cosmetic and/or nutraceutical, the method comprising the steps of:

a) contacting the target cell with a potential inhibitor of Let-7b,

b) the expression of Let-7b was quantified,

12. The method of claim 11, wherein the Let-7b inhibitor is for maintaining or improving the biomechanical properties of healthy skin and/or healthy mucous membranes, in particular for maintaining and/or improving the firmness and/or elasticity and/or density of healthy skin and/or healthy mucous membranes, in particular healthy dermis.

13. The method of any one of claims 11 and 12, wherein the Let-7b inhibitor is for maintaining or improving the organization quality of extracellular matrix, for maintaining or improving the amount of organized molecules of extracellular matrix, in particular collagen fibers and/or elastic fibers, and/or for maintaining or increasing the synthesis of glycosaminoglycans, in particular chondroitin sulfate.

14. The method of any one of claims 11 to 13, wherein the target cells are healthy human fibroblasts.

15. The method of any one of claims 11 to 14, wherein the quantifying step is a direct quantification of the amount of Let-7b and/or an indirect quantification by measuring a change in the amount of the target protein and/or its corresponding mRNA.

16. The method according to any one of claims 11 to 15, wherein the protein to be modulated and/or its corresponding mRNA is fibrillin 1(FBN1) and/or xylosyltransferase 1(XYLT1) and/or TGF β receptor 2(TGFBR2) and/or integrin β 3(ITGB3) and/or chondroitin sulphate synthase 3 (choy 3), preferably xylosyltransferase 1(XYLT1) and/or integrin β 3(ITGB3) and/or chondroitin sulphate synthase 3 (choy 3), more preferably xylosyltransferase 1(XYLT1) and/or chondroitin sulphate synthase 3 (choy 3).

17. The method of any one of claims 11 to 16, wherein the quantification of Let-7b expression is performed by direct quantification via RT-qPCR.

18. The method of any one of claims 11-17, wherein the potential inhibitor is present at a concentration ranging from 1 x 10 by weight of the Let-7b inhibitor relative to the volume of the culture medium^-4% to 10% (w/v), preferably 0.001% to 0.1% (w/v), more preferably 0.003% to 0.02% (w/v).

Cosmetic and/or nutraceutical use of a Let-7b inhibitor for maintaining or improving the biomechanical properties of healthy skin and/or healthy mucous membranes, in particular the firmness, elasticity and/or density of healthy skin and/or healthy mucous membranes, in particular healthy dermis.

20. Use according to claim 19, for maintaining or improving the organization quality of the extracellular matrix, for maintaining or increasing the amount of organized molecules of the extracellular matrix, in particular collagen fibers and/or elastic fibers, and/or for maintaining or increasing the synthesis of glycosaminoglycans, in particular chondroitin sulfate.

21. The use of claim 19 or 20, wherein the Let-7b inhibitor is selected by the method of any one of claims 11 to 18.

22. Cosmetic care method comprising the topical application of a Let-7b inhibitor and/or a cosmetic composition comprising a Let-7b inhibitor to healthy skin, in particular to healthy scalp and/or healthy mucous membranes, in order to maintain or improve the biomechanical properties of the healthy skin and/or healthy mucous membranes, in particular the firmness, elasticity and/or density of the healthy skin and/or healthy mucous membranes, in particular of the healthy dermis.

23. A cosmetic care method according to claim 22 wherein the application is carried out on all or part of the healthy body and/or face and/or scalp, preferably on the legs, thighs, arms, abdomen, neck, axilla, whole or part of the face, preferably on the cheeks and/or angular regions of the face.